(Project Leader: Noelle L'etoile) Addiction, learning deficits and cognitive decline are all mounting public health concerns. Each pathology results in a large part from aberrant neuronal plasticity. Thus, understanding the cellular and molecular basis for neuronal plasticity is of great interest. The role of extracellular RNA in neuronal plasticity has just been uncovered by our lab's unpublished work. We propose to use the powerful cell biological and genetic model organism C. elegans to discover pathways that produce and utilize exRNA and develop tools to visualize, with sub-cellular resolution, exRNAs in vivo. Mounting biochemical evidence has accumulated for exRNAs in mammals. In C. elegans the phenomenon of RNAi spreading, which requires exRNAs, is well documented. However, it is still unclear how exRNAs are produced, secreted, trafficked from one tissue type to the next, or how they alter target tissue biology. These gaps result from a paucity of tools providing high resolution, high sensitivity imaging of small RNAs in an intact organism and the lack of genetic screens for pathways required for the production and function of environmentally induced exRNAs. We have evidence that C. e/egans sensory neurons produce small RNAs in response to environmental stimulation. Though these small RNAs arise from the sensory neuron, we find they induce organism-wide increases in small RNA. This increase is dependent upon the double stranded RNA channel, SID-1. We used this observation to develop exRNA reporter strains. These will be used in screens for genes required for biogenesis, secretion, trafficking and utilization of stimulus-induced exRNAs. The stimulus forexRNA production comes from either the environment in the form of odors or from within the cell in the form of oncogene activation. This study will provide detailed genetic and cell biological insight into how endogenously triggered 22G RNAs and medically relevant microRNAs are produced and act at a distance from their source cell to affect organismal biology. The genes we identify and the imaging tools we develop in this project may provide novel mechanistic insight into the medically relevant roles of exRNA..And the irhaging tools could revolutionize small RNA cell biology by providing quantitative images of small RNAs at subcellular resolution.